N-Glycosylated Antibody

ABSTRACT

The invention relates to a monoclonal antibody or derivative or fragment thereof that is derived from a parental monoclonal antibody, that recognizes the Lewis Y antigen, characterized in that the Fc region or region equivalent to the Fc region of said antibody or derivative or fragment thereof carries a bi-sected hybrid type N-glycosylation pattern and that said antibody shows at least 10 fold increased ADCC and at least 10% reduced CDC activity.

The invention relates to a novel N-glycosylated antibody or derivativethereof carrying a bi-sected hybrid type N-glycosylation pattern andhaving increased ADCC and decreased CDC activities.

The invention also relates to the use of the antibodies for thepreparation of medicaments for the treatment of cancer.

Tumors are formed due to the unchecked cell growth which leads to theformation of solid cell agglomerates in case of epithelial cells. Incase of benign tumor tissue, it is assumed that the cell growth islimited and secondary tumors or metastases will not occur. In cancerdiseases, however, malignant tumors form, and in the progressing stagesecondary tumors and metastases occur. Most frequently, cancer formswith epithelial tumors occur which inter alia concern breast, stomach,intestines, pancreas, lungs, prostate and ovaries.

Cancer is a wide-spread disease and is lethal in many cases. The therapyof cancer usually comprises the removal of a solid tumor, and a furthertreatment which is to prevent and reduce, respectively, metastases.Besides surgery, the standard therapies include chemotherapy andradiation therapy. Despite the comprehensive therapy which ofteninvolves severe side effects, the success of treatment is insufficient.The relapse rate in intestinal cancer is approximately 45%. Metastaticepithelial cancer is considered to be nearly incurable. Therefore, inthe treatment of cancer patients it is important to prevent, and reduce,respectively, the formation of metastases.

Tumor cells are capable of disseminating from primary tumors in bodyliquids and other organs. These disseminated tumor cells may be in theirdormant state and often cannot be attacked by a chemotherapy(radiotherapy). Such a treated patient seems to be in a cured state.Dormant tumor cells, however, have a potential of forming metastases ifthey become growing and metastasizing cells, described as “minimalresidual disease”.

Immunotherapy constitutes an innovative possible treatment of cancerpatients. Both active and also passive immunotherapy are acknowledgedmeasures for supporting the immune system.

The adaptive immune system of humans consists of two essentialcomponents, the humoral and the cellular immunity. The adaptive immuneresponse partially is based on the clonal selection of BandT-lymphocytes and in principle allows for the recognition of any desiredantigen as well as for the build-up of an immunological memory. Thesecharacteristics of the adaptive immune system are generally usefullyaddressed in vaccinations.

Each B-cell produces an antibody with a defined binding specificity.This antibody is also present as a specific receptor in the membrane ofthe B-cell producing it. The humoral immune response against antigensrecognized as foreign is based on the selective activation of thoseB-cells which produce such antibodies that can bind to an epitope of therespective antigen. For the antibody diversity, DNA rearrangements inthe course of B-cell differentiation play a decisive role.

There are several possible ways of interfering in the immune system.

1. Passive Antibody Therapy

For therapeutic purposes, it is possible to supply to an organismantibodies required for a certain function within this organism. Thistype of application is called passive immunotherapy, and it can be usedin various medical indications, e.g. in the immunotherapy of cancer(Glennie M. J. and Johnson P. W. M., Immunol. Today (2000), 21:403),intoxications (Chippaux J. P. and Goyffon M., Toxicon (1998), 36:823;Sabouraud A, Scherrmann J M, Therapie (1994), 49:41) and infections(Casadevall A and Scharff M. D., Clin. Infect. Dis. (1995), 21:150). Inthese cases, antibodies can be used which either have been derived fromappropriately immunized animals or can be recovered from cells byvarious biological or molecular-biological techniques (e.g. hybridomatechnique, phage-display technique, etc.) via the immortalization ofimmunoglobulin genes.

2. Active Immunization

To modulate the immune system, an immunization with antigens can beused. Antigens are molecules, molecule complexes or whole organisms towhich antibodies can bind. Not all the antigens induce an immuneresponse, i.e. not all the antigens are immunogenic. Certain smallmolecules are not registered by the immune system (haptens), suchsmaller molecules can be presented to the immune system in suitableform, and thus be made immunogenic. Such a method is the coupling of thehapten to an immunogenic molecule, a so-called carrier molecule. For anactive immunization, also antibody preparations can be used, asdescribed in EP 1140168.

Tumor cells can be attacked by the immune system only to a limitedextent, since they are hardly different from normal cells and specificantibodies therefore are missing. Much research is directed to theidentification of suitable targets, i.e. target antigens, for thepreparation of tumor-specific antibodies. The immunotherapy for thetreatment of cancer then either comprises the passive therapy by thedirect administration of the specific antibodies, or the activevaccination with suitable antigen-targets for stimulating the immunesystem and generating the specific antibodies in vivo.

One approach of relatively specifically destroying tumor cells is thepassive immunotherapy with antibodies directed against tumor-associatedantigens (TAA) (Glennie M. J. and Johnson P. W. M., Immunology Today(2000), 21:403-410; Scott A. M. and Welt S., Curr. Opin. Immunol.(1997), 9:717).

Certain TAAs are defined as relevant “targets” for the development ofimmunotherapeutic agents for the prophylaxis and/or treatment of cancer.TAAs are structures which preferably are expressed on the cell membraneof tumor cells, thereby allow for a differentiation relative tonon-malignant tissue, and thus can be viewed as targets for thediagnostic and therapeutic applications of specific antibodies.

In the course of the discovery and the subsequent characterization ofvarious TAAs it has been found that they often have important functionsfor cancer cells. They allow the degenerate cells to have propertiescharacteristic of the malignant phenotype, such as, e.g., an increasedadhesion capacity, or an increased uptake of growth factors, which arehighly important for establishing metastases. However, in certainstages, such antigens may very well also be expressed on normal cellswhere they are responsible for normal functions of these cells. Anexample of this is the Lewis Y carbohydrate antigen which appears on theplurality of tumors of epithelial origin, but also plays an importantrole during the fetal development of epithelial tissues. It has beenshown that the expression of this antigen in lung cancer is associatedwith an unfavorable prognosis, since Lewis Y positive cancer cellsapparently have a higher metastatic potential (Miyake M. et al, N. Engl.J. Med. 327 (1992), 14).

In EP 0 528 767, the use of a humanized anti-Lewis Y antibody for thetreatment of epithelial cancer has been described.

Among the further known tumor-associated carbohydrate structures, thereare, e.g., all those Lewis antigens which are highly expressed in manytypes of epithelial cancers. Among them are Lewis x-, Lewis b- and Lewisy-structures, as well as sialylated Lewis x-structures. Othercarbohydrate antigens are Globo H-structures, KH1, Tn antigen, TFantigen, the alpha-1,3-galactosyl epitope (Curr. Pharmaceutical Design(2000), 6:485, Gollogly L. and Castronovo V., Neoplasma (1996), 43:285).

Other TAAs are proteins which are particularly highly expressed bycancer cells, such as, e.g. CEA, TAG-72, MUC1, Folate Binding ProteinA-33, CA125, EpCAM, HER-2/neu, PSA, MART, etc. (Sem. Cancer Biol.(1995), 6:321). Relevant TAAs often are surface antigens of epithelialcells which occur in larger numbers in growing cells, such as fetaltissue, and also in tumor tissue.

Direct therapeutic applications of antibodies against TAA are based onpassive immunotherapies, i.e., a specific antibody is systemicallyadministered in a suitable amount to cancer patients, and has animmunotherapeutic effect. The biological half-life of such agents willdepend on their structure and is limited. Therefore, it is necessary tocarry out repeated applications. When using xenogenic antibodies (e.g.murine monoclonal antibodies, MABs) however, this can lead to undesiredimmune reactions which may neutralize a possible therapeutic effect andmay cause dangerous side effects (anaphylactic reactions). Therefore,such immunotherapeutic agents can be administered for a limited timeonly.

A better tolerance is obtained by reducing the xenogenic structures ofthe antibody and by introducing human structures, e.g. with chimeric orhumanized antibodies. Also systems for producing specific humanantibodies are being developed. Thus, according to the prior art,certain cell lines, organisms or transgenic animals can produce humanantibodies.

Nevertheless, it is often reported that the application of high amountsof antibodies, even if these antibodies are humanized antibodies, canlead to side effects that often are dose dependent. These side effectscan be diarrhea, fever, chills, bronchospasm (immediate type allergicreaction, ITAR) etc.

It is well known that antibodies achieve their therapeutic effectthrough various mechanisms. They can have direct effects in producingapoptosis or programmed cell death. They can block growth factorreceptors, effectively arresting proliferation of tumor cells. In cellsthat express monoclonal antibodies, they can bring about anti-idiotypeantibody formation.

Indirect effects include recruiting cells that have cytotoxicity, suchas monocytes and macrophages. This type of antibody-mediated cell killis called antibody-dependent cell mediated cytotoxicity (ADCC).Monoclonal antibodies also bind complement, leading to direct celltoxicity, known as complement dependent cytotoxicity (CDC). In case ofADCC (Antibody Dependent Cell mediated Cytotoxicity), the Fc fragment ofthe monoclonal antibody binds the Fc receptors found on monocytes,macrophages, granulocytes and natural killer cells. These cells in turnengulf the bound tumor cell and destroy it. Natural killer cells secretecytokines that lead to cell death, and they also recruit B cells. Incase of CDC (Complement Dependent Cytotoxicity) the monoclonal antibodyis binding to the receptor and initiating the complement system, alsoknown as the “complement cascade”. The end result is a membrane attackcomplex that literally makes a hole within the cell membrane, causingcell lysis and death. Methods for the production of antibodies havingincreased ADCC and CDC activities have already been described (Sburlatiet al., 1998, Biotechnol. Prog., 14, 189-192; Shinkawa T. et al., 2003,J. Biol. Chem., 278, 3466-3473).

It has been shown that antibody mediated CDC activity can lead topartially severe side effects in antibody therapy. For example, it wasdescribed by van der Kolk et al. (British J. Haematol., 2001, 115,807-811) that the treatment with a chimaeric anti-CD20 antibody lead tomoderate to severe side effects which were argued due to complementactivation. It was shown that the severity of the side effectscorrelated with the level of complement activation. Especially inpatients with a high number of circulating tumor cells these sideeffects can be life threatening. The increased toxicity due tocomplement dependent cytotoxicity was suggested to be carefully examinedwhen the complement activation is enhanced.

Studies on other antibodies (OKT3) supported these observations withregard to the negative side effects induced by complement activation.

Therefore there is a great demand in providing means for antibodytherapy that avoids these unwanted side effects.

The object of the invention is to provide antibodies with improvedproperties.

According to the invention, this object is achieved by the subjectmatter of the claims.

The present inventors have generated a monoclonal antibody or derivativeor fragment thereof that is derived from a parental monoclonal antibodythat recognizes the Lewis Y antigen, characterized in that the Fc regionor region equivalent to the Fc region of said antibody or derivative orfragment thereof carries a bi-sected hybrid type N-glycosylation patternand that said antibody shows at least 10 fold increased ADCC and atleast 10% reduced CDC activity.

The parental monoclonal antibody that is directed against the Lewis Yantigen is an antibody comprising a humanized light chain variableregion, a human light chain constant region, a humanized heavy chainvariable region and a human heavy chain constant region, wherein thehumanized light chain variable region has an amino acid sequence asshown in FIG. 1 and the humanized heavy chain variable region has theamino acid sequence as shown in FIG. 2. Such antibody is described in EP0 528 767.

Preferentially the antibody according to the invention contains at leastparts of the amino acid sequence of the heavy chain variable region andheavy chain constant region of the parental antibody. Most preferred,the amino acid sequence of the inventive antibody is identical to theparental antibody. Preferably the inventive antibody is directed againstthe Lewis Y antigen.

The clinical efficacy of the parental antibody is related to thebiological activity of the Fc part of the human IgG1 molecule, which isdetermined by its efficiency in inducing antibody dependent cellularcytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).Especially the ADCC function depends on the glycosylation of the Fcpart, which interacts with the FcγRIII on granulocytes and monocytes(Lifely et al., 1995, Glycobiology, 5(8), 813-822).

Oligosaccharides normally found in the Fc region of serum IgG arecomplex bi-antennary type with low levels of terminally sialic acid andbisecting N-acetylglycosamine and a variable degree of terminalgalactosylation and core fucosylation (Lund and Takahashi, 1996, J.Immunol., 157, 4963-9). FcγR binding requires the presence ofoligosaccharides covalently attached at the conserved Asn297 in the Fcregion and is sensitive to oligosaccharide structure. IgG's expressed inhamster or mouse cell lines carry usually very similar glycosylationstructures but lack the bisecting N-acetylglucosamine (GlcNac) found inlow amounts in serum IgG.

The ADCC activity of the antibody according to the invention is at least10 fold increased, preferably at least 20 fold, more preferably at least25 fold increased ADCC activity, preferably at least 40 fold increasedADCC activity, preferably at least 60 fold increased ADCC activity, mostpreferred at least 100 fold increased ADCC activity compared to the ADCCactivity of the parental antibody.

The ADCC lysis activity of the inventive antibody can be measured incomparison to the parental antibody using six Lewis-Y positive targetcancer cell lines (SKBR5, SKBR3, LoVo, MCF7, OVCAR3 and Kato III).

According to the invention, the antibody or derivative or fragmentthereof has a CDC activity that is at least 10% decreased, or at least20% decreased, or at least 40% decreased compared to the CDC activity ofthe parental antibody.

The antibody according the invention or derivative or fragment therofcan either contain a core fucosylation or no fucosylation. It can be amurine, chimeric, human or humanized antibody, preferably the antibodyis a humanized one. In a preferred embodiment, the antibody is IgG or afragment or derivative therof, preferably IgG1 or a fragment orderivative thereof. In a further embodiment, the present inventiveantibody is a fusion protein that includes a region equivalent to the Fcregion of human IgG.

Accordingly, in one aspect the claimed invention is also directed to apharmaceutical preparation containing the antibody according to theinvention in a pharmaceutically acceptable carrier or diluent.

Furthermore, the use of this antibody as a pharmaceutical is claimed.

The pharmaceutical can be used as medicament for the prophylactic and/ortherapeutic treatment for the reduction or inhibition, respectively, ofthe growth of tumor cells in a patient, especially for the treatment ofsolid cancer, exemplary for the treatment of tumors or disseminatedtumor cells of epithelial origin. Furthermore, the antibody according tothe invention can be used for the treatment of minimal residual disease.

Terms as used herein as generally used in the art, unless otherwisedefined as follows.

The term “antibody” includes antibodies or antibody derivatives orfragments thereof. Among the antibody fragments are functionalequivalents or homologues of antibodies including any polypeptidecomprising an immunoglobulin binding domain or peptides mimicking thisbinding domain together with a Fc region or a region homologous to a Fcregion or at least part of it. Chimeric molecules comprising animmunoglobulin binding domain, or equivalents, fused to anotherpolypeptide are included. Exemplary antibody molecules are intactimmunoglobulin molecules and those portions of an immunoglobulinmolecule that contains the paratope, including those portions known asFab, Fab′, F(ab′)2,Fc and F(v).

As used herein, the antibody according to the invention can be expressedin host cells which cover any kind of cellular system which can bemodified to express the antibody. Within the scope of the invention, theterm “cells” means the cultivation of individual cells, tissues, organs,insect cells, avian cells, mammalian cells, hybridoma cells, primarycells, continuous cell lines, stem cells and/or genetically engineeredcells, such as recombinant cells expressing a glycosylated antibodyaccording to the invention.

Preferably the cells are animal cells, more preferably mammalian cells.These can be for example BSC-1 cells, LLC-MK cells, CV-1 cells, CHOcells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VEROcells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMKcells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells,BHK cells, SP2/0, NS0 cells or derivatives thereof.

The bi-sected hybrid type N-glycosylation pattern of the inventiveantibody can be produced by a glycoprotein modifiying glycosyltransferase, for example the β(1,4)-N-acetylglucosaminyltransferase(GlcNAc transferase III, GntIII) which adds the residue. The Gnt III hasalready been cloned (Miyoshi et al., 1995, J. Biol. Chem., 270:28311-28315). This can result in an antibody that carries a bi-sectingN-acetylglucosamine group in the Fc part. The glycosylation of the Fcpart of the inventive antibody can be amended by using technologies wellknown in the art. Exemplary, methods are described in Sburlati et al.,1998, Biotechnol. Prog., 14, 189- 192 or U.S. Pat. No. 6,602,684. Hereincells are capable of expressing the Gnt III activity which can increasecomplex N-linked oligosaccharides carrying bisected GlcNAc. Althoughmost of the cell lines used for production of antibodies do not containthe GntIII enzyme, there are also cells that naturally express the GntIII enzyme, for example Y0 myeloma cells or B lymphozytes.

A change in the glycosylation pattern can also be induced in host cellsby mutational techniques, for example mismatch repair system changes (WO04/09782, WO 04/24871).

A further technique for the amendment of the glycosylation pattern ofthe Fc part of the inventive antibody is described in EP 1 176 195.

The term “antibody dependent cellular cytotoxicity” (ADCC) used hereinrefers to any activity to injury a tumor cell or the like by activatingan effector cell via the binding of the Fc region of an antibody to anFc receptor existing on the surface of an effector cell such as a killercell, a natural killer cell, an activated macrophage or the like. Anantibody having increased ADCC activity can be determined by anysuitable method known by the skilled person. An accepted assay isdescribed in the examples.

Increased ADCC can be measured by an increased lytic potential measuredas a decreased EC50 antibody concentration which indicates the antibodyconcentration necessary to specifically lyse the half-maximal amount oftarget cells.

The term “complement dependent cytotoxicity” (CDC) is defined as directcell toxicity by binding and activation of complement. An antibody isbinding to its target on the cell surface of e.g. the tumor cell andinitiates the complement system, also known as “complement cascade”resulting in a membrane attack complex that literally makes a holewithin the cell membrane, causing cell lysis and death. An antibodyhaving decreased CDC activity can be determined by any suitable methodknown by the skilled person. An accepted assay is described in theexamples.

Decreased CDC activity can be defined as an increased EC50 antibodyconcentration which enables the lysis of the half-maximal amount oftarget cells.

Core fucosylation of an N-linked oligo-saccharide means the presence ofa Fucose group linked α-1,6 linked to the Asn neighboring Glc-Nac group.

The binding activity of the inventive antibody to the Lewis y antigen isat least 80% compared to the parental antibody, preferentially at least90%, more preferentially 100%.

The assembling of constant and light chains of the inventiveglycosylated antibody is similar to the parental antibody.

Surprisingly, it was also shown that the N-linked antibody glycosylationpattern according to the invention shows higher homogeneity due to adecreased amount of found oligo-saccharide structures compared to theparental antibody. This leads to the preferred effect that variationbetween newly produced batches of the antibody is reduced and thereforea more stable and more homogenous antibody preparation is obtained.

A possible treatment objective is the effective binding and reduction oftumor cells, i.e. tumor tissue or metastases or, in particular,disseminated tumor cells. The number of tumor cells, or micrometastases,respectively, detectable in blood, bone marrow or organs shall besignificantly reduced. The formation of metastases is to be retarded,their growth is at least to be slowed down. Thus, the relapse-free lifespan and thus also the total survival time of the patients can belengthened by the specifically targeted immunotherapy.

Within the scope of the use according to the invention, in particularthe treatment for reducing, or inhibiting, respectively, the growth oftumor cells in a cancer patient, also a hemodialysis is possible.

For binding all the glycosylated receptors of a tumor cell, usually ahigh doses of at least 50 mg/dose, preferably at least 100 mg/dose, mostpreferred at least 200 mg/dose per patient is administered. The maximumdose will depend on the tolerability of the antibody, humanizedantibodies, and human antibodies, respectively, being best tolerated. Adose of up to 1 g or in some instances up to 2 g per patient andtreatment may very well be advantageous.

Surprisingly, it has been shown in the present invention that due to theincreased ADCC activity the amount of antibody as applied fortherapeutic and/or prophylactic purpose can be reduced, yet stillleading to positive therapeutic effects even in reduced doses. Due tothe increased ADCC activity the amount of antibody applied can bereduced at least 10%, preferably at least 20%, more preferably at least30%, most preferably at least 50% compared to the dosage regimen for theparental antibody.

Alternatively, the antibody according to the invention can be applied invery high dosages. This is based on the fact that the inventive antibodyhas reduced CDC activity which is known to cause side effects inindividuals. Therefore especially in risk patients who have developedalready side effects as a result of application of antibodies forpurposes of passive immunotherapy, this surprisingly positive propertycan be of high advantage.

The treatment preferably is repeated at certain time intervals,according to the half life of the antibody used, which usually is in therange of from 3 to 30 days. By particularly derivatizing the antibody itis possible to increase the half life to up to several months and tothereby lengthen the treatment intervals accordingly. The medicamentused according to the invention preferably is provided in a suitableformulation. Preferred are such formulations with a pharmaceuticallyacceptable carrier. The latter comprises, e.g., auxiliary agents,buffers, salts and preservatives. Preferably, a ready to use infusionsolution is provided. Since an antibody is relatively stable,medicaments based on antibodies or their derivatives have thesubstantial advantage that they can be put on the market as astorage-stable solution, or as a formulation in a ready-to-use form. Theformer preferably is storage-stable in the formulation at refrigeratortemperatures up to room temperature. The medicament used according tothe invention may, however, also be provided in frozen or lyophilizedform which may be thawed or reconstituted when required.

The concentration of the active substance of the medicament will dependon its tolerability. A particularly well tolerable preparation based ona humanized antibody can be administered directly to the patient at ahigh concentration without further dilution. By the preferredconcentration in the range of from 0.1% to 10%, preferably from 1% to5%, it is possible to keep low the administered volume and thecorresponding time of infusion. Usually, the medicament will beadministered i.v. Likewise, however, also another parenteral or mucosalmode of administration can be chosen, which brings the active substanceto a systemic or local application at the site of the tumor or of themetastases.

EXAMPLES

The following examples shall explain the present invention in moredetail, without, however, restricting it.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Sequences of the humanized light chain variable region.

FIG. 2: Sequence of the humanized heavy chain variable region. Eithersequence 1 or 2 can be used.

FIG. 3: Binding activity analysis of IGN311 Glyco I compared to IGN311wt.

FIG. 4: Results of CDC analysis using SKBR 5 cells as target

FIG. 5: Results of the ADCC lysis experiments of IGN312 Glyco I onSKBR3, Ovcar 3, Kato III and A421 cells.

FIG. 6: Schematic picture of a bi-sected hybrid type N-glycosylatedantibody. N-acetylglucosamines are depicted as squares, mannoses aredepicted as circles, galactoses are depicted as ellipses.

Example 1 Abbreviations SEC-HPLC Size Exclusion Chromatography—HighPerformance Liquid Chromatography PBS Phosphate Buffered Saline SDS-PAGESodium-Lauryl-Sulfate-Poly-Acrylamide-Gel-Electrophoresis ADCC Antibodydependent cellular cytotoxicity CDC Complement dependent cytotoxicityIGN311 humanized monoclonal anti-Lewis Y antibody IGN312Glyco-engineered humanized monoclonal anti-Lewis Y antibody withidentical specificity as IGN311 ELISA Enzyme linked immuno sorbant assayMethods SDS-PAGE

Integrity, size and potential degradation products of purifiedexpression product were analyzed by SDS-PAGE. Samples were diluted in4×NuPAGE SDS sample buffer and incubated at 85° C. for 10 min. 10 μlwere loaded on Novex NuPAGE 4-12% Bis-Tris gels (Invitrogen) run in aNovex Electrophoresis unit for 50 min at 200 V and 125 mA. Gels weresilver-stained according to the instructions of the manufacturer(Invitrogen).

IEF (isoelectric focusing)

Isoform-distribution, degradation and potential deamidation productswere analyzed by IEF. After dilution in Novex IEF sample buffer(Invitrogen), samples were loaded on a Novex IEF pH 3-10 Gel andseparated according to the instructions of the manufacturer. Gels weresilver-stained.

ELISA Based on Anti-idiotypic Antibody Specific for IGN311

Binding activity of the expression product was analyzed by a specificsandwich ELISA by incubating antibody samples in serial dilutions (from100 pg to 1 μg/ml) in microtiter wells coated with the monoclonalanti-idiotypic antibody MMA383. After blocking with 3% FCS and washing,bound expression product was determined by incubation with goatanti-human IgG+A+M/Peroxidase conjugate (Zymed, Calif.) and developedwith o-phenylenediamine/hydrogen peroxide. Measurements were made usingan ELISA reader at 492 nm/620 nm. Measured optical densities wereplotted versus logarithm of the antibody concentration (ng/ml) andfitted using a sigmoidal four parameter fit. EC50 values were calculatedand used for quantification.

CDC

The complement mediated lytic activity was tested in a ⁵¹Cr releaseassay using the Le-Y antigen-positive SKBR5 breast cancer cell line astarget cells. Target cells were incubated for one hour with 100 μCi of⁵¹Cr, washed twice with medium and plated at a density of 20×10³ cellsper well into a 96-well microtiter plate together with a serial dilutionof the sample to be analyzed (100 ng to 50 μg/ml) and complement serumfrom a volunteer donor. The test plate was incubated for 1 hour at 37°C. in a CO₂ incubator. Supernatants were collected and counted forreleased ⁵¹Cr (Cs). Values for spontaneous release (Sr) and maximumrelease (Mr) were measured after incubation of representative sampleswith medium alone or with detergent (SDS), respectively. Complementmediated cytotoxicity was calculated as percentage of cell lysis100×(Cs-Sr)/(Mr-Sr). The percent cytotoxicity was plotted versuslogarithm of the antibody concentration (ng/ml) and fitted using asigmoidal four parameter fit. EC50 values were calculated and used forquantification.

ADCC

The cellular mediated lytic potential was tested in a ⁵¹Cr release assayusing different Le-Y antigen-positive cancer cell lines as target cells(SKBR3, Kato III and Ovcar 3). Target cells were incubated for one hourwith 100 μCi of ⁵¹Cr, washed, and plated at a density of 25×10³ cellsper well into 96-well mictrotiter plate. Effector cells (peripheralblood mononucleocytes from a volunteer donor) were freshly prepared andadded to the target cells to achieve E:T ratios of 40:1 together withserial dilutions (100 pg to 1 μg/ml) of the antibody sample to beanalyzed. After incubation at 37° C. for 18 hours in a CO₂ incubator,cell supernatants were collected and counted for released ⁵¹Cr (Cs).Values for spontaneous release (Sr) and maximum release (Mr) weremeasured after incubation of representative samples with medium alone orwith detergent (SDS) respectively. Cytotoxicity was calculated aspercentage of cell lysis 100×(Cs-Sr)/(Mr-Sr). The percent cytotoxicitywas plotted versus logarithm of the antibody concentration (ng/ml) andfitted using a sigmoidal four parameter fit. EC50 values were calculatedand used for quantification.

The antibody producing cell line was genetically modified in order toexpress the glycosyl transferase Gnt-III in order to enhance thebiological activity of the antibody. The modification was according tothe techniques as described in Sburlati et al (Biotechnol. Prog., 1998,14, 189-192) or U.S. Pat. No 6,602,684.

In a first setup, heavy and light chain genes of IGN311 were isolated,cloned into an expression vector and transfected transiently into EBNAcells: Genes for Gnt-III transferase expression were co-transfectedresulting in a new antibody called IGN312. A control wild-type antibodyIGN311 wt. was expressed using exactly the same expression vectors andthe same host but without co-transfection of genes for Gnt-IIIexpression. Both expression products were purified to homogenicity usingan identical Protein-A based down stream process. Expression productswere characterized by SDS-PAGE, IEF and a target antigen specificsandwich ELISA. No degradation products could be detected and targetaffinity of the glyco-engineered antibody as well as assembling of heavyand light chains was not affected by Gnt-III expression.

Results: Analysis of the Glycosylated Antibody (IGN312)

Glyco-engineered expression product IGN312 (IGN312 Glyco I) was comparedto IGN311 wt. By SDS-PAGE analysis. Under non-reducing conditions, bothproteins showed exactly the same bands in the range of nearly 150 kDacorresponding to the expected molecular weight of an intact IgG. Underreducing conditions, protein bands of nearly 50 and 25 kDa could bestained corresponding to IgG heavy and light chains, respectively. Nodifferences between the expression products could be found. Nodegradation products or aggregates were detected. The glyco-engineeredversion of IGN311 was an intact and correctly assembled IgG.

Comparison of IGN312 and IGN311 wt. In isoelectric focusing analysisshowed exactly the same band distribution between a pI of 7.8 and 8.3.Four protein bands of different pI and different amount could bevisualized.

FIG. 3 shows the binding activity analysis of IGN311 Glyco I (blackcurve) compared to IGN311 wt. (grey curve). Data series were fittedusing a four parameter sigmoidal fit.

Antibody specificity of the glyco-engineered product was analyzed by itsantigen binding activity in an anti-idiotypic ELISA. Dilution curves aredisplayed graphically in FIG. 4. All curves showed exactly the sameshape and could be overlaid. The same assertion can be made by comparingvalues of sigmoid curve fit and evaluation at EC50. Results of a sigmoidfour parameter fit of data series are shown in Table 1. Normalization ofactivities on IGN311 wt. resulted in very similar values. No significantchanges in affinity could be detected therefore and antigen binding ofglyco-engineered product was maintained in the range of the original,not glyco-engineered product.

TABLE 1 Binding activity of IGN312 Glyco I measured by an anti-idiotypic binding ELISA, results of a four parameter sigmoid fit. IGN311wt. IGN312 Glyco I Bottom 96.12 77.04 Top 2063 2105 Log EC50 1.805 1.838Hillslope −1.376 −1.208 EC50 63.76 ng/ml 68.81 ng/ml % 100 108

Analysis of effector functions were studied in vitro using threedifferent Lewis-Y positive tumor cell lines as target cells. Expressionof the target glycosylation pattern Le-Y was investigated prior lysisexperiments by FACS analysis using IGN311 wt. as detection antibody.SKBR 3 showed the most intense Le-Y expression followed by Ovcar 3 andfinally Kato III (geometric mean fluorescence: SKBR 3: 1803, Ovcar 3:361, Kato III: 55). A421 has lost its Le-Y positivity and was usedtherefore as negative control cell line in our experimental setup. Lysespotential of the glyco-engineered antibody via cellular cytotoxicity andvia complement activation was analyzed.

FIG. 4 shows results of CDC analysis using SKBR 5 cells as target. Dataseries were fitted using a sigmoid four parameter fit (Table 2).Evaluation was performed at EC50 and lyses potential was normalized onthe activity of IGN311 wt. Lyses potential of IGN312 Glyco I viacomplement activation appeared to be nearly 44 % reduced. FIG. 4 showsthe CDC analysis (Chromium release) on SKBR5 target cells. Complementdependent cytotoxicity of IGN312 Glyco I (black curve) and IGN311 wt.(grey curve) were compared. Data series were fitted (Sigmoid fourparameter fit).

TABLE 2 CDC lysis activity of IGN312 Glyco I measured by a chromiumrelease assay using SKBR5 as target cell line. Data values were fittedusing a four parameter sigmoid fit. IGN311 wt. IGN312 Glyco I Bottom−0.747 2.217 Top 70.92 70.13 Log EC50 2.743 2.998 Hillslope 3.281 2.834EC50 553.1 ng/ml 996.2 ng/ml % 100 56

Results of lyses potential analysis of IGN312 via antibody dependentcellular cytotoxicity (ADCC) was analyzed on SKBR 3, Ovcar 3, Kato IIIand A421 cells. Results are displayed in FIG. 4. Data series were fittedusing a sigmoid four parameter model. Lyses activity via ADCC wascalculated by normalization on the activity of IGN311 wt. and evaluationat EC50. Results are displayed in Table 3. Lysis activity of IGN312Glyco I was significantly enhanced in all cases from 6 to 14 fold incomparison to IGN311 wt. A direct correlation between Lewis-Y antigendensity measured by FACS analysis (geometric mean fluorescence: SKBR3:1803, Ovcar3: 361, Kato III: 55) and enhanced cellular cytotoxicitycould not be found. A421 showed, as expected, no lysis at all.

TABLE 3 ADCC analysis on different Lewis-Y positive target cells.Antibody dependent cellular cytotoxicity of IGN312 Glyco I and IGN311wt. were compared. Data series were fitted using a sigmoidal fourparameter fit. Activity was calculated by normalization on IGN311 wt.IGN311 wt. IGN312 Glyco I SKBR3 Bottom −0.6914 4.473 Top 97.25 96.28 LogEC50 1.694 0.8728 Hillslope 1.019 1.459 EC50 49.4 ng/ml 7.46 ng/ml % 100662 Kato III Bottom −0.3125 −2.374 Top 68.1 77.74 Log EC50 3.063 2.164Hillslope 1.019 0.9021 EC50 1157 ng/ml 145.9 ng/ml % 100 793 Ovar 3Bottom 0.0679 −14.68 Top 84.78 94.65 Log EC50 1.94 0.8887 Hillslope1.202 0.8948 EC50 87.15 ng/ml 7.74 ng/ml % 100 1126 A421 Bottom 0 0 Top0 0 Log EC50 0 0 Hillslope 0 0 EC50 0 0 % 0 0

FIG. 5 shows results of the ADCC lysis experiments of IGN312 Glyco I onSKBR3, Ovcar 3, Kato III and A421 cells.

Discussion and Conclusion

The data presented in this study indicate that it is possible to enhancethe ADCC activity of an IgG1 antibody while lowering the CDC capacity byglyco-engineering. This study showed that genetic engineering of theglycosylation apparatus of an industrial expression cell line can be avery interesting tool for the modulation and fine tuning of the effectorfunctions of an over-expressed antibody. Antibody dependent cellularcytotoxicity could be significantly increased up to at least 20 fold bythis principle. The amount of mannosylated hybrid-oligo-saccharidestructures increased in parallel which decreased complement activation.

A balanced and stable expression level on Gnt-III transferase wouldtherefore be an essential requirement for the generation of aglyco-engineered therapeutic antibody with enhanced lytic potential. Theestablishment of a production process based on a stable IGN312expressing cell line will be the next crucial step in order to finallycompare clinically the efficiency of this “new generation antibody”.

Example 2 Lysis of Disseminated Tumor Cells Using the Parental AntibodyIGN311—Clinical Reports Dosage and Duration of Administration

This is a multiple-treatment, escalating dosage study. All subjectsreceived IGN311 on Day 1 and 15. The first three evaluable subjectsreceived 50 mg IGN311 per infusion, the next three evaluable subjectsreceived 100 mg IGN311 per infusion and the last three evaluablesubjects received 200 mg IGN311 per infusion.

IGN311 was administered intravenously in a slow infusion during a twohour period.

The subject should not receive any other therapies for the treatment ofcarcinoma (e.g. chemotherapeutic, radiation, immune therapy or anyinvestigational agent other than IGN311) during the study period withthe exceptions of bisphosphonates and hormonal therapy.

Assessment of Disseminated Tumor Cells in Peripheral Blood: SampleCollection

Blood samples were collected on Day 1 and Day 15 before start ofinfusion and on Day 43. Blood samples (approximately 28 ml) werecollected by a single venous puncture (from the antecubital vein orother suitable site as determined by the investigator) intoanticoagulation collection tubes (Vacutainer® tube with EDTA). To avoidthe contamination of the blood samples with epithelial cells accumulatedduring the penetration of the needle through the skin, the first 3 mlblood had to be collected in a separate Vacutainer and had to bediscarded. The tumor cell enrichment protocol had to be processed within2 hours.

Enrichment of Tumor Cells

25 ml cooled peripheral blood were gently filled into the uppercompartment of an OncoQuick® tube without disturbing the separationmedium underneath the porous barrier and centrifuged at 1600×g and 4° C.for 20 min. After centrifugation, the interphase between the upperplasma (yellow/brownish) and the lower separation medium (blue),containing the tumor cells, were collected and transferred into a new 50ml centrifugation tube and washed. The cell pellet was then beresuspended in 4 ml washing buffer and centrifuged onto microscopeslides.

Results:

It has been shown that the number of tumor cells which had been detectedin several sear from patients decreased due to the treatment withIGN311.

1. Monoclonal antibody or derivative or fragment thereof that is derived from a parental monoclonal antibody that recognizes the Lewis Y antigen, characterized in that the Fc region or region equivalent to the Fc region of said antibody or derivative or fragment thereof carries a bi-sected hybrid type N-glycosylation pattern and that said antibody shows at least 10 fold increased ADCC and at least 10% reduced CDC activity.
 2. Antibody according to claim 1, characterized in that the ADCC activity is increased at least 20 fold.
 3. Antibody according to claim 1, characterized in that the ADCC activity is increased at least 25 fold.
 4. Antibody according to claim 1, characterized in that the ADCC activity is increased at least 40 fold.
 5. Antibody according to claim 1, characterized in that the ADCC activity is increased at least 60 fold.
 6. Antibody according to claim 1, characterized in that the CDC activity is at least 20% decreased.
 7. Antibody according to claim 1, characterized in that the CDC activity is at least 40% decreased.
 8. Antibody according to any one of claims 1 to 7, characterized in that it carries a bi-secting N-acetylglucosamine group.
 9. Antibody according to any one of claims 1 to 8, characterized in that said antibody does not contain a core fucosylation.
 10. Antibody according to any one of claims 1 to 9, characterized in that it is a humanized antibody.
 11. Antibody according to any one of claims 1 to 10 which is IgG or a fragment or derivative thereof.
 12. Antibody according to claim 11 which is IgG1 or a fragment or derivative thereof.
 13. Pharmaceutical preparation containing an antibody according to any one of claims 1 to 12 in a pharmaceutically acceptable carrier or diluent.
 14. Use of an antibody according to any one of claims 1 to 12 as a pharmaceutical.
 15. Use of a preparation based on an antibody according to any one of claims 1 to 12 for preparing a medicament for the prophylactic and/or therapeutic treatment for the reduction or inhibition, respectively, of the growth of tumor cells in a patient.
 16. Use of an antibody according to any one of claims 1 to 12 for the manufacture of a medicament for the treatment of solid cancer.
 17. Use to claim 15 for the treatment of solid cancer that is of epithelial origin.
 18. Use according to claim 15 for treating the “minimal residual disease”.
 19. Use according to claim 14 for passive immunotherapy.
 20. Use according to any one of claims 1 to 12, characterized in that said antibody is used in a dosage of at least 50 mg/dose.
 21. Use according to any one of claims 1 to 12, characterized in that said antibody is used in a dosage of at least 100 mg/dose.
 22. Use according to any one of claims 1 to 12, characterized in that said antibody is used in a dosage of at least 200 mg/dose. 